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1.
Genes Dev ; 25(21): 2306-20, 2011 Nov 01.
Article in English | MEDLINE | ID: mdl-22056673

ABSTRACT

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.


Subject(s)
Proteome , Proteomics/methods , Animals , Bacterial Proteins/genetics , Databases, Protein , Embryo, Nonmammalian , Genetic Vectors , Internet , Luminescent Proteins/genetics , Molecular Sequence Annotation , Mutation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Zebrafish
2.
Dev Cell ; 18(6): 973-84, 2010 Jun 15.
Article in English | MEDLINE | ID: mdl-20627079

ABSTRACT

Extracellular matrix (ECM) remodeling is critical for organogenesis, yet its molecular regulation is poorly understood. In zebrafish, asymmetric migration of the epithelial lateral plate mesoderm (LPM) displaces the gut leftward, allowing correct placement of the liver and pancreas. To observe LPM migration at cellular resolution, we transgenically expressed EGFP under the control of the regulatory sequences of the bHLH transcription factor gene hand2. We found that laminin is distributed along the LPM/gut boundary during gut looping, and that it appears to become diminished by the migrating hand2-expressing cells. Laminin diminishment is necessary for LPM migration and is dependent on matrix metalloproteinase (MMP) activity. Loss of Hand2 function causes reduced MMP activity and prolonged laminin deposition at the LPM/gut boundary, leading to failed asymmetric LPM migration and gut looping. Our study reveals an unexpected role for Hand2, a key regulator of cell specification and differentiation, in modulating ECM remodeling during organogenesis.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Extracellular Matrix/metabolism , Gastrointestinal Tract/embryology , Gastrointestinal Tract/metabolism , Organogenesis/physiology , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Animals, Genetically Modified , Basic Helix-Loop-Helix Transcription Factors/genetics , Body Patterning/physiology , Cell Differentiation/physiology , Cell Lineage/physiology , Cell Movement/physiology , Gastrointestinal Tract/cytology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Laminin/metabolism , Matrix Metalloproteinases/metabolism , Mesoderm/cytology , Mesoderm/metabolism , Transgenes/genetics , Zebrafish Proteins/genetics
3.
Dev Biol ; 277(1): 1-15, 2005 Jan 01.
Article in English | MEDLINE | ID: mdl-15572135

ABSTRACT

Identification of cardiac mechanisms of retinoic acid (RA) signaling, description of homologous genetic circuits in Ciona intestinalis and consolidation of views on the secondary heart field have fundamental, but still unrecognized implications for vertebrate heart evolution. Utilizing concepts from evolution, development, zoology, and circulatory physiology, we evaluate the strengths of animal models and scenarios for the origin of vertebrate hearts. Analyzing chordates, lower and higher vertebrates, we propose a paradigm picturing vertebrate hearts as advanced circulatory pumps formed by segments, chambered or not, devoted to inflow or outflow. We suggest that chambers arose not as single units, but as components of a peristaltic pump divided by patterning events, contrasting with scenarios assuming that chambers developed one at a time. Recognizing RA signaling as a potential mechanism patterning cardiac segments, we propose to use it as a tool to scrutinize the phylogenetic origins of cardiac chambers within chordates. Finally, we integrate recent ideas on cardiac development such as the ballooning and secondary/anterior heart field paradigms, showing how inflow/outflow patterning may interact with developmental mechanisms suggested by these models.


Subject(s)
Biological Evolution , Heart/anatomy & histology , Heart/embryology , Animals , Humans , Phylogeny , Signal Transduction , Tretinoin/pharmacology
4.
Development ; 130(22): 5363-74, 2003 Nov.
Article in English | MEDLINE | ID: mdl-13129847

ABSTRACT

Establishment of anteroposterior (AP) polarity is one of the earliest decisions in cardiogenesis and plays an important role in the coupling between heart and blood vessels. Recent research implicated retinoic acid (RA) in the communication of AP polarity to the heart. We utilized embryo culture, in situ hybridization, morphometry, fate mapping and treatment with the RA pan-antagonist BMS493 to investigate the relationship between cardiac precursors and RA signalling. We describe two phases of AP signalling by RA, reflected in RALDH2 expression. The first phase (HH4-7) is characterized by increasing proximity between sino-atrial precursors and the lateral mesoderm expressing RALDH2. In this phase, RA signalling is consistent with diffusion of the morphogen from a large field rather than a single hot spot. The second phase (HH7-8) is characterized by progressive encircling of cardiac precursors by a field of RALDH2 originating from a dynamic and evolutionary-conserved caudorostral wave pattern in the lateral mesoderm. At this phase, cardiac AP patterning by RA is consistent with localized action of RA by regulated activation of the Raldh2 gene within an embryonic domain. Systemic treatment with BMS493 altered the cardiac fate map such that ventricular precursors were found in areas normally devoid of them. Topical application of BMS493 inhibited atrial differentiation in left anterior lateral mesoderm. Identification of the caudorostral wave of RALDH2 as the endogenous source of RA establishing cardiac AP fates provides a useful model to approach the mechanisms whereby the vertebrate embryo confers axial information on its organs.


Subject(s)
Aldehyde Oxidoreductases/metabolism , Body Patterning/physiology , Heart/embryology , Animals , Chick Embryo , Embryonic Induction/physiology , Mesoderm/enzymology , Mice , Retinal Dehydrogenase
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